Furin-knockdown and GM-CSF-augmented (FANG) cancer vaccine

ABSTRACT

Compositions and methods for cancer treatment are disclosed herein. More specifically, the present invention describes an autologous cancer vaccine genetically modified for Furin knockdown and GM-CSF expression. The vaccine described herein attenuates the immunosuppressive activity of TGF-β through the use of bi-functional shRNAs to knock down the expression of furin in cancer cells, and to augment tumor antigen expression, presentation, and processing through expression of the GM-CSF transgene.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation of application Ser. No. 14/815,721, filed Jul.31, 2015, now issued as U.S. Pat. No. 9,790,518 on Oct. 17, 2017, whichis a continuation of application Ser. No. 12/973,823, filed on Dec. 20,2010, now issued as U.S. Pat. No. 9,132,146 on Sep. 15, 2015, and claimsbenefit of priority to U.S. Provisional Application No. 61/289,681,filed Dec. 23, 2009, and U.S. Provisional Application No. 61/309,777,filed Mar. 2, 2010, the contents of each are incorporated herein byreference in their entireties.

TECHNICAL FIELD OF THE INVENTION

The present invention relates in general to the field of vaccinedevelopment, and more particularly, to the development of compositionsand methods for making and using an autologous cancer vaccinegenetically modified for Furin knockdown and GM-CSF expression.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created Jul. 5, 2018, isnamed 51867706302_SL.txt and is 2.221 bytes in size.

BACKGROUND OF THE INVENTION

Without limiting the scope of the invention, its background is describedin connection with the development of genetically modified whole cellcancer vaccines. More specifically, the present invention relates tovaccines capable of augmenting tumor antigen expression, presentation,and processing through expression of the GM-CSF transgene andattenuating secretory immunosuppressive activity of TGF-β via furinbi-functional shRNA transgene induced knockdown.

The prevailing hypothesis for immune tolerance to cancer vaccinesinclude the low immunogenicity of the tumor cells, the lack ofappropriate presentation by professional antigen presenting cells,immune selection of antigen-loss variants, tumor inducedimmunosuppression, and tumor induced privileged site. Whole cancer cellvaccines can potentially solicit broad-based, polyvalent immuneresponses to both defined and undefined tumor antigens, therebyaddressing the possibility of tumor resistance through downregulationand/or selection for antigen-loss variants. A method for making a mastercell bank of whole cell vaccines for the treatment of cancer can befound in U.S. Pat. No. 7,763,461 issued to Link et al. (2010). Accordingto the '461 patent, tumor cells are engineered to express an α (1,3)galactosyl epitope through ex-vivo gene therapy protocols. The cells arethen irradiated or otherwise killed and administered to a patient. Theα-galactosyl epitope causes opsonization of the tumor cell enhancinguptake of the opsonized tumor cell by antigen presenting cells whichresults in enhanced tumor specific antigen presentation. The animal'simmune system thus is stimulated to produce tumor specific cytotoxiccells and antibodies which will attack and kill tumor cells present inthe animal.

Granulocyte-macrophage colony-stimulating factor, often abbreviated toGM-CSF, is a protein secreted by macrophages, T cells, mast cells,endothelial cells and fibroblasts. When integrated as a cytokinetransgene, GM-CSF enhances presentation of cancer vaccine peptides,tumor cell lysates, or whole tumor cells from either autologous patienttumor cells or established allogeneic tumor cell lines. GM-CSF inducesthe differentiation of hematopoietic precursors and attracts them to thesite of vaccination. GM-CSF also functions as an adjuvant for dendriticcell maturation and activation processes. However, GM-CSF-mediatedimmunosensitization can be suppressed by different isoforms oftransforming growth factor beta (TGF-β) produced and/or secreted bytumor cells. The TGF-β family of multifunctional proteins possesses wellknown immunosuppressive activities. The three known TGF-β ligands(TGF-β1, -β2, and -β3) are ubiquitous in human cancers. TGF-βoverexpression correlates with tumor progression and poor prognosis.Elevated TGF-β levels within the tumor microenvironment are linked to ananergic tumor response. TGF-β directly and indirectly inhibits GM-CSFinduced maturation of dendritic cells and their expression of MHC classII and co-stimulatory molecules. This negative impact of TGF-β onGM-CSF-mediated immune activation supports the rationale of depletingTGF-β secretion in GM-CSF-based cancer cell vaccines.

All mature isoforms of TGF-β require furin-mediated limited proteolyticcleavage for proper activity. Furin, a calcium-dependent serineendoprotease, is a member of the subtilisin-like proprotein convertasefamily. Furin is best known for the functional activation of TGF-β withcorresponding immunoregulatory ramifications. Apart from the previouslydescribed immunosuppressive activities of tumor secreted TGF-β,conditional deletion of endogenously expressed furin in T lymphocyteshas been found to allow for normal T-cell development, but impairedfunction of regulatory and effector T cells, which produced less TGF-β1.Furin expression by T cells appears to be indispensable in maintainingperipheral tolerance, which is due, at least in part, to itsnon-redundant, essential function in regulating TGF-β1 production.

High levels of furin have been demonstrated in virtually all cancerlines. The inventors and others have found that up to a 10-fold higherlevel of TGF-β1 may be produced by human colorectal, lung cancer, andmelanoma cells, and likely impact the immune tolerance state by a highermagnitude. The presence of furin in tumor cells likely contributessignificantly to the maintenance of tumor directed, TGF-β-mediatedperipheral immune tolerance. Hence furin knockdown represents a noveland attractive approach for optimizing GM-CSF-mediatedimmunosensitization and vaccine development. Chen et al. (2004) in U.S.patent application Ser. No. 20040242518 provide methods and compositionsfor inhibiting influenza infection and/or replication based on thephenomenon of RNAi as well as systems for identifying effective siRNAsand shRNAs for inhibiting influenza virus and systems for studyinginfluenza virus infective mechanisms. The invention also providesmethods and compositions for inhibiting infection, pathogenicity and/orreplication of other infectious agents, particularly those that infectcells that are directly accessible from outside the body, e.g., skincells or mucosal cells. In addition, the invention provides compositionscomprising an RNAi-inducing entity, e.g., an siRNA, shRNA, orRNAi-inducing vector targeted to an influenza virus transcript and anyof a variety of delivery agents. The invention further includes methodsof use of the compositions for treatment of influenza.

Interferon-gamma (γIFN) is a key immunoregulatory cytokine that plays acritical role in the host innate and adaptive immune response and intumor control. Also known as type II interferon, γIFN is a single-copygene whose expression is regulated at multiple levels. γIFN coordinatesa diverse array of cellular programs through transcriptional regulationof immunologically relevant genes. Initially, it was believed that CD4+T helper cell type 1 (Th1) lymphocytes, CD8+ cytotoxic lymphocytes, andNK cells exclusively produced γIFN. However, there is now evidence thatother cells, such as B cells, NKT cells, and professionalantigen-presenting cells (APCs) secrete γIFN. γIFN production byprofessional APCs [monocyte/macrophage, dendritic cells (DCs)] actinglocally may be important in cell self-activation and activation ofnearby cells. γIFN secretion by NK cells and possibly professional APCsis likely to be important in early host defense against infection,whereas T lymphocytes become the major source of γIFN in the adaptiveimmune response. Furthermore, a role for γIFN in preventing developmentof primary and transplanted tumors has been identified. γIFN productionis controlled by cytokines secreted by APCs, most notably interleukins(IL) IL-12 and IL-18. Negative regulators of γIFN production includeIL-4, IL-10, glucocorticoids, and TGF-β.

SUMMARY OF THE INVENTION

The present invention also provides an autologous (i.e., patientspecific) cancer vaccine composition (FANG vaccine), comprising atherapeutically effective amount of cells with an shRNA^(furin)/GM-CSFexpression vector. This vector comprises a first nucleic acid encodingGM-CSF, which may be human GM-CSF, and a second nucleic acid insertencoding one or more short hairpin RNAs (shRNA) capable of hybridizingto a region of an mRNA transcript encoding furin, thereby inhibitingfurin expression via RNA interference. Both nucleic acid inserts areoperably linked to a promoter. The shRNA may be bi-functional,incorporating both (cleavage dependent) RISC (RNA induced silencingcomplex) formatted) siRNA (cleavage dependent) and (cleavage-independentRISC formatted) either miRNA or miRNA-like motifs simultaneously. In oneembodiment of the present invention, the shRNA is both the RISC cleavagedependent and RISC cleavage independent inhibitor of furin expression.Furthermore, the expression vector may contain a picornaviral 2Aribosomal skip peptide intercalated between the first and the secondnucleic acid inserts, and the promoter may be CMV mammalian promoterwhich could contain an enhancer sequence and intron. The mRNA sequencestargeted by the bi-functional shRNA are not limited to coding sequences;in one embodiment, the shRNA may target the 3′ untranslated region(3′-UTR) sequence of the furin mRNA transcript, and in one embodimentmay target both the coding sequence and the 3′ UTR sequence of the furinmRNA transcript simultaneously. The cells used to produce the vaccinemay be autologous tumor cells, but xenograft expanded autologous tumorcells, allogeneic tumor cells, xenograft expanded allogeneic tumorcells, or combinations of them may also be used. The vaccine dosageadministered to patients contains 1×10⁷ cells to 2.5×10⁷ cells. The FANGvaccine can be given in conjunction with a therapeutically effectiveamount of γIFN (gamma interferon). The dosage range of γIFN may be 50 or100 μg/m².

The present invention describes an autologous cell vaccine compositioncomprising: a bi-shRNA^(furin)/GM-CSF expression vector plasmid and oneor more optional vaccine adjuvants. The vector plasmid comprises a firstnucleic acid insert operably linked to a promoter, wherein the firstinsert encodes a Granulocyte Macrophage Colony Stimulating Factor(GM-CSF) cDNA and a second nucleic acid insert operably linked to thepromoter, wherein the second insert encodes one or more short hairpinRNAs (shRNA) capable of hybridizing to a region of a mRNA transcriptencoding furin, thereby inhibiting furin expression via RNAinterference. In one aspect the GM-CSF is human. In another aspect theshRNA incorporates siRNA (cleavage dependent RISC formatted) and eithermiRNA or miRNA-like (cleavage-independent RISC formatted) motifs. TheshRNA as described herein is both the cleavage dependent RISC formattedand cleavage independent RISC formatted inhibitor of furin expressionand is further defined as a bi-functional shRNA.

In another aspect, a picornaviral 2A ribosomal skip peptide isintercalated between the first and the second nucleic acid inserts. Inyet another aspect the promoter is a CMV mammalian promoter containing aCMV IE 5′ UTR enhancer sequence and a CMV IE Intron A. In other aspectsthe region targeted by the shRNA is the 3′ UTR region sequence of thefurin mRNA transcript and the region targeted by the shRNA is the codingregion of the furin mRNA transcript.

The present invention provides a method of preventing, treating and/orameliorating symptoms of a cancer in a patient by comprising the stepsof: identifying the patient in need of prevention, treatment, and/oramelioration of the symptoms of the cancer and administering anautologous cell vaccine comprising a bi-shRNA^(furin)/GM-CSF expressionvector plasmid, wherein the vector plasmid comprises a first nucleicacid insert operably linked to a promoter, wherein the first insertencodes a Granulocyte Macrophage Colony Stimulating Factor (GM-CSF)cDNA, a second nucleic acid insert operably linked to the promoter,wherein the second insert encodes one or more short hairpin RNAs (shRNA)capable of hybridizing to a region of a mRNA transcript encoding furin,thereby inhibiting furin expression via RNA interference, and one ormore optional vaccine adjuvants.

The method further comprises the steps of monitoring the progression ofthe therapy by measuring a level of a transforming growth factor beta(TGF-beta or TGF-β) and the GM-CSF in the one or more cancer cells,wherein a reduction in the level of TGF-β and an elevation in the levelof the GM-CSF is indicative of a successful therapy and altering theadministration of the autologous cell vaccine based on the levels of theTGF-β and the GM-CSF. As per the method of the present invention theTGF-β is selected from at least one of TGF-β1, TGF-β2, or TGF-β3. In oneaspect the cancer is selected from the group consisting of melanoma,non-small cell lung cancer, gall bladder cancer, colorectal cancer,breast cancer, ovarian, liver cancer, liver cancer metastases, andEwing's sarcoma as well as other patient derived TGF-β producingcancers. In another aspect the shRNA incorporates siRNA (cleavagedependent RISC formatted) and either miRNA or miRNA-like(cleavage-independent RISC formatted) motifs and the shRNA is both thecleavage dependent RISC formatted and cleavage independent RISCformatted inhibitor of furin expression. In yet another aspect the shRNAis further defined as a bi-functional shRNA.

In another embodiment the present invention discloses an autologousfurin-knockdown and Granulocyte Macrophage Colony Stimulating Factor(GM-CSF) augmented (FANG) cancer vaccine composition comprising: abi-shRNA^(furin)/GM-CSF expression vector plasmid, wherein the vectorplasmid comprises a first nucleic acid insert operably linked to apromoter, wherein the first insert encodes the GM-CSF cDNA and a secondnucleic acid insert operably linked to the promoter, wherein the secondinsert encodes one or more short hairpin RNAs (shRNA) capable ofhybridizing to a region of a mRNA transcript encoding furin, therebyinhibiting furin expression via RNA interference and one or moreoptional vaccine adjuvants. The composition of the present invention isused to prevent, treat, and/or ameliorate the symptoms of a cancer,wherein the cancer is selected from the group consisting of melanoma,non-small cell lung cancer, gall bladder cancer, colorectal cancer,breast cancer, ovarian, liver cancer, liver cancer metastases, andEwing's sarcoma as well as other patient derived TGF-β producingcancers.

In yet another embodiment the present invention is a method of treating,preventing, and/or ameliorating the symptoms of a non-small cell lungcancer (NSCLC) in a patient by an administration of a furin-knockdownand Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) augmented(FANG) cancer vaccine comprising the steps of: identifying the patientin need of prevention, treatment, and/or amelioration of the symptoms ofthe NSCLC and administering the FANG vaccine comprising abi-shRNA^(furin)/GM-CSF expression vector plasmid, wherein the vectorplasmid comprises a first nucleic acid insert operably linked to apromoter, wherein the first insert encodes the GM-CSF cDNA, a secondnucleic acid insert operably linked to the promoter, wherein the secondinsert encodes one or more short hairpin RNAs (shRNA) capable ofhybridizing to a region of a mRNA transcript encoding furin, therebyinhibiting furin expression via RNA interference, and one or moreoptional vaccine adjuvants. The method of the instant invention furthercomprising the steps of: monitoring the progression of the therapy bymeasuring a level of a transforming growth factor beta (TGF-beta orTGF-β) and the GM-CSF in the one or more NSCLC cells, wherein areduction in the level of TGF-β and an elevation in the level of theGM-CSF is indicative of a successful therapy and altering theadministration of the autologous cell vaccine based on the levels of theTGF-β and the GM-CSF. In one aspect of the present invention the TGF-βis selected from at least one of TGF-β1, TGF-β2, or TGF-β3.

The present invention in a further embodiment describes a method ofmaking a furin-knockdown and Granulocyte Macrophage Colony StimulatingFactor (GM-CSF) augmented (FANG) cancer vaccine comprising the steps of:(i) harvesting one or more cancer cells from a patient aseptically, (ii)placing the harvested cells in an antibiotic solution in a sterilecontainer, (iii) forming a cell suspension from the harvested solution,wherein the formation of the cell, (iv) suspension is achieved byenzymatic dissection, mechanical disaggregation or both, (v) modifyingthe cells genetically by electroporating the cell suspension to make thevaccine with a bi-shRNA^(furin)/GM-CSF expression vector plasmid,wherein the vector plasmid comprises a first nucleic acid insertoperably linked to a promoter, wherein the first insert encodes theGM-CSF cDNA, a second nucleic acid insert operably linked to thepromoter, wherein the second insert encodes one or more short hairpinRNAs (shRNA) capable of hybridizing to a region of a mRNA transcriptencoding furin, thereby inhibiting furin expression via RNAinterference, (vi) harvesting the vaccine, (vii) irradiating the vaccineand (viii) freezing the vaccine.

In one aspect of the method the one or more cancer cells are harvestedfrom a patient suffering from a cancer selected from the groupconsisting of melanoma, non-small-cell lung cancer, gall bladder cancer,colorectal cancer, breast cancer, ovarian, liver cancer, liver cancermetastases, and Ewing's sarcoma as well as other patient derived TGF-βproducing cancers. In another aspect the genetically modified cells havebeen rendered proliferation-incompetent by irradiation. In yet anotheraspect the genetically modified cells are autologous, allogenic, orxenograft expanded cells.

In one aspect the allogenic cells are established cell lines. In anotheraspect the genetically modified cells are administered to the subjectonce a month for up to 12 doses, wherein the dose of geneticallymodified cells administered to the subject is 1×10⁷ cells/injection to5×10⁷ cells/injection and the administration of the genetically modifiedcells is part of a combination therapy with an additional therapeuticagent. In yet another aspect the additional therapeutic agent used inthe combination therapy is γIFN, wherein the dose of γIFN administeredto the subject in the combination therapy is 50 or 100 μg/m². The methodof the present invention further comprises the step of incubating thegenetically modified cells with γIFN after transfection, wherein thedose of γIFN applied to the genetically modified cells aftertransfection is approximately 250 U/ml (500 U/ml over 24 hours to 100U/ml over 48 hours).

Another embodiment of the invention is a siRNA-mediated method toinhibit the expression of transforming growth factor beta (TGF-β) viafurin knockdown. This method comprises the steps of selecting a targetcell and transfecting the target cell with an expression vectorcomprising a promoter and a nucleic acid insert operably linked to thepromoter. The insert encodes one or more short hairpin RNAs (shRNA)capable of hybridizing to a region of an mRNA transcript encoding furin,consequently inhibiting furin expression via RNA interference. The shRNAmay be bi-functional, i.e., it may simultaneously incorporate siRNA(cleavage dependent RISC formatted) and either miRNA or miRNA-like(cleavage-independent RISC formatted) motifs, and inhibit furinexpression in both a cleavage dependent RISC formatted and cleavageindependent RISC formatted manner. Additionally, the expression vectormay target the coding region of the furin mRNA transcript, or it maytarget the 3′ UTR region sequence of the furin mRNA transcript, or itmay target both the coding sequence and the 3′ UTR sequence of the furinmRNA transcript simultaneously.

The present invention also provides a method to augment antigenexpression, presentation, and processing, and to attenuate secretoryimmunosuppressive activity of transforming growth factor beta (TGF-betaor TGF-β) in target cells. This method comprises the steps of selectinga target cell and transfecting the target cell with an expression vectorcomprising two inserts. The technique used to transfect the target cellsmay be plasmid vector electroporation. The first nucleic acid insertencodes GM-CSF, whereas the second insert encodes one or more shorthairpin RNAs (shRNAs) capable of hybridizing to a region of an mRNAtranscript encoding furin, thereby inhibiting furin expression via RNAinterference. Both inserts are operably linked to a promoter. The TGF-βisoforms whose activation would be precluded by knocking down furinexpression include TGF-β1, TGF-β2, and TGF-β3. Target cells may includeautologous or allogeneic cells, which may be established human celllines.

The present invention also includes a method of preventing, treatingand/or ameliorating symptoms of cancer by administering the FANG vaccineto patients. This method comprises the steps of: (i) identifying asubject in need of treatment; (ii) harvesting a cancer tissue samplefrom the subject; (iii) genetically modifying the cancer cells in theharvested cancer sample; and (iv) administering a therapeuticallyeffective dose of genetically modified cells to the subject. Theexpression vector used to transfect the cells comprises two nucleic acidinserts. The first nucleic acid insert encodes GM-CSF and it is operablylinked to a promoter. The second nucleic acid insert is also operablylinked to the promoter, and it encodes one or more short hairpin RNAs(shRNAs) capable of hybridizing to a region of an mRNA transcriptencoding furin, thereby inhibiting furin expression via RNAinterference. In one embodiment of the present invention, the cancertargeted for treatment is a human melanoma or a non-small cell lungcancer. To render the genetically modified cellsproliferation-incompetent, they may be irradiated. The geneticallymodified cells in the FANG vaccine may be autologous cells, allogeneiccells, xenograft expanded cells, established human cell lines, orcombinations of these cellular types. For vaccination, cells areadministered to the subject once a month for up to 12 doses, each onecontaining 1×10⁷ cells to 2.5×10⁷ cells. Dose escalation to 5×10⁷ hasbeen shown to be safe.

The genetically modified cells can be administered as a stand-alonetherapy; however, they may also be administered as part of a combinationtherapy. In this embodiment of the invention, the FANG vaccine may becombined with another therapeutic agent[s], such as, but not limited to,IL-12, IL-15 and/or γIFN. When including γIFN in the treatment with FANGvaccine, the method comprises the further step of incubating thegenetically modified cells with approximately 100 U/ml of γIFN for 48hours or 500 U/ml for 24 hours, respectively, after transfection. Forcombination therapy, cells are administered to the subject once a monthfor up to 12 doses, each one containing typically 1×10⁷ cells to 2.5×10⁷cells (although doses up to 5×10⁷ have been shown to be safe) plus adose of γIFN of 50 or 100 μg/m². The method further comprises the stepof incubating the genetically modified cells with γIFN aftertransfection, wherein the dose of γIFN applied to the geneticallymodified cells after transfection is approximately 250 U/ml.

The present invention includes a unique method of inhibiting TGF-βthrough RNA interference with furin, a proprotein convertase involvedcritically in the functional processing of all TGF-β isoforms. The FANGvector uniquely incorporates a bi-functional small hairpin construct(shRNA^(furin)) specific for the knockdown of furin. The bi-functionalshRNA^(furin) of the present invention comprises a two stem-loopstructure with a miR-30a backbone. The first stem-loop structure is thesiRNA precursor component, while the second stem-loop structure is themiRNA-like precursor component. In this embodiment, the strategy is touse a single targeted site for both cleavage and sequestering mechanismsof RNA interference. In one embodiment, the strategy is to use twodifferent targeted sites, one for the cleavage and one for thesequestering component comprised of, but not limited to, the codingregion of the mRNA transcript and the 3′ UTR region of the mRNAtranscript, respectively. In this embodiment, the bi-functionalshRNA^(furin) is comprised of two stem-loop structures with miR-30abackbone; the first stem-loop structure has complete complementaryguiding strand and passenger strand, while the second stem-loopstructure has three basepair (bp) mismatches at positions 9 to 11 of thepassenger strand. In one embodiment, the bi-functional shRNA^(furin) iscomprised of two stem-loop structures with miR-30a backbone; the firststem-loop structure has complete complementary guiding strand andpassenger strand, while the second stem-loop structure has threebasepair (bp) mismatches at positions 9 to 11 of the guide strand. Inother embodiments, basepair (bp) mismatches will occupy positionspreventing Ago 2 mediated cleavage and make it thermodynamicallyfavorable for passenger strand departure. In other embodiments thebasepair mismatches will occupy positions of the guide strand. The FANGconstruct contains GM-CSF and the bi-functional shRNA^(furin)transcripts under the control of a mammalian promoter (CMV) that drivesthe entire cassette. This construct is used to generate an autologous(i.e., patient specific) cancer vaccine genetically modified for furinknockdown and GM-CSF expression.

The construct used to produce the FANG vaccine in the present inventionincludes a bi-functional shRNA^(furin)/GM-CSF expression vector plasmidcomprising two nucleic acid inserts. The first nucleic acid insert islinked operably to a promoter, and it encodes a Granulocyte MacrophageColony Stimulating Factor (GM-CSF) cDNA. The second nucleic acid insertis also linked operably to the promoter, and it encodes one or moreshort hairpin RNAs (shRNA) capable of hybridizing to a region of an mRNAtranscript encoding furin, thereby inhibiting furin expression via RNAinterference. The bi-functional shRNA of the present invention has twomechanistic pathways of action, that of the siRNA and that of the miRNA.Thus, the bi-functional shRNA of the present invention is different froma traditional shRNA, i.e., a DNA transcription derived RNA acting by thesiRNA mechanism of action or from a “doublet shRNA” that refers to twoshRNAs, each acting against the expression of two different genes but inthe traditional siRNA mode. In one embodiment of the invention, theGM-CSF is human. The shRNA is bi-functional, incorporating both siRNA(cleavage dependent RISC formatted) and either miRNA or miRNA-like(cleavage-independent RISC formatted) motifs simultaneously. In oneembodiment of the present invention, the shRNA is both the cleavagedependent RISC formatted and cleavage independent RISC formattedinhibitor of furin expression. The expression vector may contain apicornaviral 2A ribosomal skip peptide intercalated between the firstand the second nucleic acid inserts, and the promoter may be CMVmammalian promoter which could contain a CMV IE 5′ UTR enhancer sequenceand a CMV IE Intron A. The mRNA sequences targeted by the bi-functionalshRNA are not limited to coding sequences; in one embodiment, the shRNAmay target the 3′ untranslated region (UTR) sequence of the furin mRNAtranscript and, in one embodiment, target both the coding sequence andthe 3′ UTR sequence of the furin mRNA transcript simultaneously.

The present invention also includes a vector that may be used tospecifically knock down the expression of furin in target cells. ThisshRNA^(furin) expression vector comprises a nucleic acid insert linkedoperably to a promoter. Such insert encodes one or more short hairpinRNAs (shRNA) capable of hybridizing to a region of an mRNA transcriptencoding furin, thereby inhibiting furin expression via RNAinterference. The bi-functional shRNA may simultaneously incorporatesiRNA (cleavage dependent RISC formatted) and either miRNA ormi-RNA-like (cleavage-independent RISC formatted) motifs, and inhibitfurin expression in both a cleavage dependent RISC formatted andcleavage independent RISC formatted manner. Additionally, the expressionvector may target the coding region of the furin mRNA transcript, or itmay target the 3′ UTR region sequence of the furin mRNA transcript, orit may target both the coding sequence and the 3′ UTR sequence of thefurin mRNA transcript simultaneously.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the features and advantages of thepresent invention, reference is now made to the detailed description ofthe invention along with the accompanying figures and in which:

FIGS. 1A-1C are plots showing the summary of: (1A) TGF-β1, (1B) TGF-β2,and (1C) GM-CSF protein production pre and post FANG plasmidtransfection. ELISA values from Day 4 of the 14-day determinations ofcytokine production in manufactured autologous cancer cells. Datarepresents autologous vaccines independently generated from 10 patientswho underwent FANG processing (FANG 001-010).

FIGS. 2A-2F are plots showing: (2A) TGF-β1, (2B) TGF-β2, and (2C) GM-CSFexpression in FANG-004 tumor cells pre and post FANG cGMP plasmidtransfection and (2D) TGF-β1, (2E) TGF-β2, and (2F) GM-CSF expression inTAG-004 tumor cells pre and post TAG cGMP plasmid transfection. FANG-004and TAG-004 are from the same tumor and processed sequentially on thesame two days as a demonstration of comparative expression profiles.

FIGS. 3A-3C are plots showing that the side-by-side comparison ofelectroporation of FANG plasmid (the cGMP vaccine manufacturing process)versus the TAG plasmid into patient tumor cells demonstrated (3C) GM-CSFprotein production and concomitantly; (3A) TGF-β1 expression knockeddown by FANG but not TAG and (3B) TGF-β2 knockdown by both FANG and TAG(coincident line).

FIG. 4A is a schematic showing the bi-shRNA^(furin) (SEQ ID NO: 2)comprising two stem-loop structures with miR-30a backbone (SEQ ID NO.:1); the first stem-loop structure has complete complementary guidingstrand and passenger strand, while the second stem-loop structure hasthree basepair (bp) mismatches at positions 9 to 11 of the passengerstrand.

FIG. 4B shows the siRNA targeted regions of furin mRNA. ProspectivesiRNA targeting regions in 3′-UTR and encoding regions of furin mRNA andthe targeted sequence by each siRNA.

FIG. 5 shows a PET CT after 11 cycles of TAG treatment in a patientdemonstrating significant response. Residual uptake at L 2 was followedup with a MRI scan and biopsy which revealed no malignancy.

FIGS. 6A-6C show an assessment of GM-CSF expression and TGF-β1 andTGF-β2 knockdown, summarizing: (6A) TGF-β1, (6B) TGF-β2, and (6C) GM-CSFprotein production before and after FANG or TAG (TAG 004) plasmidtransfection. Values represent ELISA determinations of cytokineproduction in harvested autologous cancer cells transfected with FANG.Data represents autologous vaccines independently generated from 9patients who underwent FANG processing (FANG 001-009). One patient hadsufficient tissue to construct both a FANG and TAG vaccine (FANG 004/TAG004).

FIG. 7 shows the GM-CSF mRNA by RT-qPCR in pre and post FANGtransfected/irradiated tumor cells. Absent bands in some of the lanes isdue to degraded RNA. (The extra band in FANG-009 is Day 0 sample loadedtwice).

FIG. 8 shows FANG Vaccine cells from a patient pre-transfection andpost-transfection mRNA by PCR. No signal was detected in pre- andpost-samples for TGF-β2.

FIG. 9 shows FANG-009 Vaccine cells pre-transfection andpost-transfection mRNA by PCR.

FIG. 10 shows the overall survival for Cohort 1 versus Cohorts 2 and 3for advanced-stage patients (n=61; P=0.0186).

FIG. 11 shows a schematic diagram of GM-CSF TGF-β2 antisense (TAG)plasmid.

FIG. 12 shows the expression of GM-CSF in NCI-H-460 Squamous Cell andNCI-H-520, Large Cell (NSCLC) containing the pUMVC3-GM-CSF-2A-TGF-β2antisense vector, in vitro.

FIG. 13 shows that TGF-β2 levels are reduced in NCI-H-460 Squamous Celland NCI-H-520, Large Cell (NSCLC) with the pUMVC3-GM-CSF-2A-TGF-β2antisense (TAG) vector.

FIG. 14 shows that a 251 base pair probe specifically detects theGM-CSF-2A-TGF-β2 (TAG) transcript expressed in vitro in NCI-H-460 andNCI-H-520 cells (lanes 6 and 10).

FIG. 15 shows the GM-CSF expression in TAG vaccines.

FIG. 16 shows the TGF-β1 expression in TAG vaccines.

FIG. 17 shows the TGF-β2 expression in TAG vaccines.

FIGS. 18A and 18B show expression of: (18A) TGF-β1 and (18B) TGF-β2 inhuman cancer lines following siRNA^(furin) knockdown

FIG. 19 shows the plasmid construct of FANG.

DETAILED DESCRIPTION OF THE INVENTION

While the making and using of various embodiments of the presentinvention are discussed in detail below, it should be appreciated thatthe present invention provides many applicable inventive concepts thatcan be embodied in a wide variety of specific contexts. The specificembodiments discussed herein are merely illustrative of specific ways tomake and use the invention and do not delimit the scope of theinvention.

To facilitate the understanding of this invention, a number of terms aredefined below. Terms defined herein have meanings as commonly understoodby a person of ordinary skill in the areas relevant to the presentinvention. Terms such as “a”, “an” and “the” are not intended to referto only a singular entity, but include the general class of which aspecific example may be used for illustration. The terminology herein isused to describe specific embodiments of the invention, but their usagedoes not delimit the invention, except as outlined in the claims.

It is contemplated that any embodiment discussed in this specificationcan be implemented with respect to any method, kit, reagent, orcomposition of the invention, and vice versa. Furthermore, compositionsof the invention can be used to achieve methods of the invention.

As used herein the term “nucleic acid” or “nucleic acid molecule” refersto polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleicacid (RNA), oligonucleotides, fragments generated by the polymerasechain reaction (PCR), and fragments generated by any of ligation,scission, endonuclease action, and exonuclease action. Nucleic acidmolecules can be composed of monomers that are naturally-occurringnucleotides (such as DNA and RNA), or analogs of naturally-occurringnucleotides (e.g., α-enantiomeric forms of naturally-occurringnucleotides), or a combination of both. Modified nucleotides can havealterations in sugar moieties and/or in pyrimidine or purine basemoieties. Sugar modifications include, for example, replacement of oneor more hydroxyl groups with halogens, alkyl groups, amines, and azidogroups, or sugars can be functionalized as ethers or esters. Moreover,the entire sugar moiety can be replaced with sterically andelectronically similar structures, such as aza-sugars and carbocyclicsugar analogs. Examples of modifications in a base moiety includealkylated purines and pyrimidines, acylated purines or pyrimidines, orother well-known heterocyclic substitutes. Nucleic acid monomers can belinked by phosphodiester bonds or analogs of such linkages. Analogs ofphosphodiester linkages include phosphorothioate, phosphorodithioate,phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate,phosphoranilidate, phosphoramidate, and the like. The term “nucleic acidmolecule” also includes so-called “peptide nucleic acids,” whichcomprise naturally-occurring or modified nucleic acid bases attached toa polyamide backbone. Nucleic acids can be either single stranded ordouble stranded.

The term “expression vector” as used herein in the specification and theclaims includes nucleic acid molecules encoding a gene that is expressedin a host cell. Typically, an expression vector comprises atranscription promoter, a gene, and a transcription terminator. Geneexpression is usually placed under the control of a promoter, and such agene is said to be “operably linked to” the promoter. Similarly, aregulatory element and a core promoter are operably linked if theregulatory element modulates the activity of the core promoter. The term“promoter” refers to any DNA sequence which, when associated with astructural gene in a host yeast cell, increases, for that structuralgene, one or more of 1) transcription, 2) translation or 3) mRNAstability, compared to transcription, translation or mRNA stability(longer half-life of mRNA) in the absence of the promoter sequence,under appropriate growth conditions.

The term “oncogene” as used herein refers to genes that permit theformation and survival of malignant neoplastic cells (Bradshaw, T. K.:Mutagenesis 1, 91-97 (1986)).

As used herein the term “receptor” denotes a cell-associated proteinthat binds to a bioactive molecule termed a “ligand.” This interactionmediates the effect of the ligand on the cell. Receptors can be membranebound, cytosolic or nuclear; monomeric (e.g., thyroid stimulatinghormone receptor, beta-adrenergic receptor) or multimeric (e.g., PDGFreceptor, growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSFreceptor, erythropoietin receptor and IL-6 receptor). Membrane-boundreceptors are characterized by a multi-domain structure comprising anextracellular ligand-binding domain and an intracellular effector domainthat is typically involved in signal transduction. In certainmembrane-bound receptors, the extracellular ligand-binding domain andthe intracellular effector domain are located in separate polypeptidesthat comprise the complete functional receptor.

The term “hybridizing” refers to any process by which a strand ofnucleic acid binds with a complementary strand through base pairing.

The term “transfection” refers to the introduction of foreign DNA intoeukaryotic cells. Transfection may be accomplished by a variety of meansknown to the art including, e.g., calcium phosphate-DNAco-precipitation, DEAE-dextran-mediated transfection, polybrene-mediatedtransfection, electroporation, microinjection, liposome fusion,lipofection, protoplast fusion, retroviral infection, and biolistics.

As used herein, the term “liposome” refers to a closed structurecomposed of lipid bilayers surrounding an internal aqueous space. Theterm “polycation” as used herein denotes a material having multiplecationic moieties, such as quaternary ammonium radicals, in the samemolecule and includes the free bases as well as thepharmaceutically-acceptable salts thereof.

TABLE 1 Abbreviations Table Abbreviation Term AE Adverse event ALTAlanine transaminase (also referred to as SGPT) ANC Absolute neutrophilcount APC Antigen Presenting Cells AST Aspartate transaminase (alsoreferred to as SGOT) BUN Blood urea nitrogen CBC Complete blood count CDCluster of differentiation CMV Cytomegalovirus CO₂ Total carbon dioxideCR Complete response CRF Case report form CTCAE Common Toxicity Criteriafor Adverse Events CTL Cytotoxic T lymphocyte DC Dendritic cell(s) DTHDelayed-type hypersensitivity ECOG PS Eastern Cooperative Oncology GroupPerformance Score ELISA Enzyme-Linked ImmunoSorbent Assay ELISPOTEnzyme-Linked ImmunoSorbent Spot ER Endoplasmic reticulum FANGbi-shRNA^(furin) and GM-CSF Augmented Autologous Tumor Cell Vaccine FLFlt-3-Ligand GM-CSF Granulocyte Macrophage-Colony Stimulating FactorFactor (Accession No. NM_000758) GMP Good manufacturing practice GVAXGM-CSF Secreting autologous or allogenic tumor cells HLA Human LeukocyteAntigen IBC Institutional Biosafety Committee IEC Independent EthicsCommittee IL Infiltrating lymphocytes IRB Institutional Review Board LAKLymphokine-activated killer LD Longest diameter LLC Large latent complexMHC Major histocompatability complex MLR Mixed lymphocyte reaction MRMannose receptor NK Natural Killer NKT Natural Killer T cell(s) NSCLCNon-small cell lung cancer PCR Polymerase chain reaction PD Progressivedisease PR Partial response PS Performance Status RECIST ResponseEvaluation Criteria in Solid Tumors SCLC Small cell lung cancer SDStable disease SLC Small latent complex STMN1 Stathmin 1 TAP transporterassociated with Ag processing TGF-β Transforming growth factor-β TILTumor infiltrating lymphocytes TNF Tumor necrosis factor ULN Upperlimits of normal WNL Within normal limits

Furin is a member of the subtilisin-like proprotein convertase family.The members of this family are proprotein convertases (PCs) that processlatent precursor proteins into their biologically active products.Furin, a calcium-dependent serine endoprotease, efficiently cleavesprecursor proteins at their paired basic amino acid processing sites bythe consensus sequence -Arg-X-K/Arg-Arg (RXK/RR), (SEQ ID NO: 6), with-RXXR- (SEQ ID NO: 1) constituting the minimal cleavage site. Like manyother proteases, PCs are synthesized as inactive zymogens with anN-terminal prosegment extension, which is autocatalytically removed inthe endoplasmic reticulum to achieve functionality.

High levels of furin have been demonstrated in virtually all cancerlines. (Furin, Accession No. NM_002569). A 10-fold higher level ofTGF-β1 may be produced by human colorectal, lung cancer and melanomacells, and likely impact the immune tolerance state by a highermagnitude. Transforming growth factors betas (TGF-β) are a family ofmultifunctional proteins with well-known immunosuppressive activities.The three known TGF-β ligands (TGF-β1, TGF-β2, and TGF-β3 Accession Nos.NM_000660, NM_003238, NM_003239.2, respectively) are ubiquitous in humancancers. TGF-β overexpression correlates with tumor progression and poorprognosis. Elevated TGF-β levels within the tumor microenvironment arelinked to an anergic antitumor response. The presence of furin in tumorcells likely contributes significantly to the maintenance of tumordirected TGF-β1 mediated peripheral immune tolerance. Hence, furinknockdown represents a novel and attractive approach for optimizingimmunosensitization.

A Furin-knockdown and GM-CSF-augmented (FANG) Autologous Cancer Vaccinefor Human Melanoma and Lung Cancer: FANG uniquely incorporates abi-functional small hairpin RNA (shRNA) construct specific for theknockdown of furin, a proprotein convertase critically involved in thefunctional processing of all TGF-β isoforms. Prior work by the inventorshas demonstrated the effectiveness of FANG in generating GM-CSFexpression and TGF-β1 and TGF-β2 depletion in human cancer lines. Theincorporation of a bi-functional shRNA^(furin) in combination withhGM-CSF into an autologous cell vaccine is demonstrated herein topromote and enhance the immune response based on its effect on theafferent limb of that immune response.

As used herein the term “bi-functional” refers to a shRNA having twomechanistic pathways of action, that of the siRNA and that of the miRNA(the guide strand being non-complementary to the mRNA transcript) ormiRNA-like (the guide strand being complementary to the mRNAtranscript). The term “traditional” shRNA refers to a DNA transcriptionderived RNA acting by the siRNA mechanism of action. The term “doublet”shRNA refers to two shRNAs, each acting against the expression of twodifferent genes but in the “traditional” siRNA mode.

Survival of patients with advanced NSCLC, the most common cancerinvolving both men and women, is 7 months or less following treatmentwith second line chemotherapy. Limited survival benefit and toxicityrelated to the cancer and the treatment commonly forces patients todecline further therapy. Demonstration of safety and extensive clinicaljustification including examples of dramatic response related to“targeted” immune stimulation and suppression of endogenous immuneinhibition using the novel, mature technology of the present inventiondescribed herein provides an opportunity for safe and potentiallyeffective clinical assessment. The commercial expansion of the RNAinterference technology and vaccine manufacturing of the presentinvention will provide a gateway opportunity into management of NSCLCand likely other solid tumors, notably melanoma, ovary, prostate cancer,colon cancer and breast cancer.

Overcoming immune tolerance with cancer vaccines is a promising butdifficult quest. The prevailing hypotheses for immune tolerance, basedprimarily on animal studies, include the low immunogenicity of the tumorcells, the lack of appropriate presentation by professional antigenpresenting cells, immune selection of antigen-loss tumor variants, tumorinduced immunosuppression, and tumor-induced privileged site [1].Nevertheless, recent clinical trials that are based ontransgene-expressing whole cancer cell vaccines have yielded promisingresults [2-5]. Whole cancer cell vaccines can potentially elicitbroad-based, polyvalent immune responses to both defined and undefinedtumor antigens, thereby addressing the possibility of tumor resistancethrough downregulation and/or selection for antigen-loss variants [6,7].

Dranoff and Jaffee have shown in animal models [8], that tumor cellsgenetically modified to secrete GM-CSF, as compared to other cytokines,consistently demonstrated the most potent induction of anti-tumorimmunity. When integrated as a cytokine transgene, GM-CSF enhancespresentation of cancer vaccine peptides, tumor cell lysates, or wholetumor cells from either autologous or established allogeneic tumor celllines [9]. GM-CSF induces the differentiation of hematopoieticprecursors into professional antigen presenting (APC) dendritic cells(DC) and attracts them to the site of vaccination [8,10]. GM-CSF alsofunctions as an adjuvant for the DC maturation and activationalprocesses of tumor antigen capture, process and presentation,upregulates their expression of costimulatory molecules, and theirability to migrate to secondary lymphoid tissues for activation of CD4+,CD8+ T cells, CD1d restricted invariant natural killer T (NKT) cells,and antibody producing B cells [11].

Recently, Hodi [12] reported that GV AX vaccination, followed byperiodic infusions of anti-CTLA-4 antibodies to modulate effector and Tregulatory cell functions, can generate clinically meaningful antitumorimmunity in a majority of metastatic melanoma patients. These findingsare consistent with the thesis that vaccination with a GM-CSF-augmentedautologous cancer vaccine can successfully generate an immune mediatedtumor destruction, particularly when coupled with an adjuvant treatmentthat depletes FoxP3+ Tregs activity, enhances tumor expression of MHCclass I A chain (MICA) thereby activating natural killer (NK) and Tcells, and enhances central memory T-cell CD4+ and CD8+ response.

The FANG approach of the present invention is supported by the findingsof the inventors in 10 patients' autologous vaccines, which demonstratedconsistently TGF-β1 and TGF-β2 reductions and elevated GM-CSF levels(FIGS. 1A-1C and FIGS. 2A-2F). Soundness of the furin-depletion approachhas been confirmed by proof of principle documentation with the furininhibitor decanoyl-Arg-Val-Lys-Arg-CMK (SEQ ID NO: 3) (Dec-RVKR-CMK)(SEQ ID NO: 3) in cancer cell lines (CCL-247 colorectal, CRL-1585melanoma lines). Dec-RVKR-CMK (SEQ ID NO: 3) is a peptidylchloromethylketone that binds irreversibly to the catalytic site offurin and blocks its activity [59]. Dec-RVKR-CMK (SEQ ID NO: 3) eithercompletely or partially reduces the activity of furin substrates BASE((3-site APP-cleaving enzyme), MT5-MMP, and Boc-RVRR-AMC (SEQ ID NO: 4)[60]. The present inventors found both TGF-β1 and TGF-β2 activity to besignificantly reduced in CCL-247 and CRL-1585 cancer lines by specificimmunoassay, confirming the effectiveness of furin blockade on TGF-βisoform expression.

The FANG plasmid (FIG. 19) used to transfect the autologous cells isderived from the TAG plasmid [74] by replacing the human TGF-β2antisense sequence with the bi-shRNA^(furin) DNA sequence. Otherwisethese two plasmids are identical (confirmed by DNA sequencing). Thebi-shRNA^(furin) consists of two stem-loop structures with miR-30abackbone; the first stem-loop structure has complete complementaryguiding strand and passenger strand, while the second stem-loopstructure has three bp mismatches at positions 9 to 11 of the passengerstrand (FIG. 4A). The inventors adopted a strategy of using a singletargeted site for both cleavage and sequestration. The encoding shRNA isable to accommodate mature shRNA loaded onto more than one types of RISC[65]. The rationale for focusing on a single site is that multi-sitetargeting may increase the chance for “seed sequence” induced off-targeteffect [66]. The two stem-loop double stranded DNA sequence wasassembled with 10 pieces of synthetic complementing and interconnectingoligonucleotides through DNA ligation. The completed 241 base pairs DNAwith BamHI sites at both ends was inserted into the BamHI site of theTAG expression vector in place of the TGF-β2 antisense sequence.Orientation of the inserted DNA was screened by PCR primer pairsdesigned to screen for the shRNA insert and orientation. The FANGconstruct has a single mammalian promoter (CMV) that drives the entirecassette, with an intervening 2A ribosomal skip peptide between theGM-CSF and the furin bi-functional shRNA transcript, followed by arabbit polyA tail. There is a stop codon at the end of the GM-CSFtranscript. Insertion of picornaviral2A sequences into mRNAs causesribosomes to skip formation of a peptide bond at the junction of the 2Aand downstream sequence, leading to the production of two proteins froma single open reading frame [67]. However, in the instances in whichshRNA or antisense are being expressed as the second transcript (asexamples), only the first transcript is translated. The inventors foundthat the 2A linker to be effective for generating approximately equallevels of GM-CSF and anti-TFG-β transcripts with the TAG vaccine, andelected to use the same design for FANG.

The inventors validated the applicability of siRNA-mediatedfurin-knockdown for inhibiting human TGF-β isoform expression.Prospective siRNA targeting sites (FIG. 4B) in the furin mRNA sequencewere determined by the published recommendations of Tusch1 andcolleagues and the additional selection parameters that integrates BLASTsearches of the human and mouse genome databases [73]. siRNAs targetingeligible coding and 3′ UTRs sites (FIG. 4B) were tested. Followinglipofection of CCL-247, CRL-1585 U87 and H460 cells, each of the 6siRNA^(furin) constructs was shown to markedly reduce TGF-β1 and TGF-β2levels in culture supernatants without adversely affecting cellsurvival. Thus siRNA-mediated furin knockdown is effective for thedepletion of TGF-β1 and -β2 isoforms.

The present inventors attempted to detect endogenous Furin protein incell lines via Western Blot and Flow Cytometry. Five differentcommercial antibodies were screened for Western Blot and one pre-labeledantibody was screened for Flow Cytometry. All studies yielded negativeresults. Upon further study of the commercially available antibodies,all idiotypes were developed against fragments (or peptides) of theFurin protein. The Western Blot studies demonstrated that the 60 kDavariant was preferentially detected in 4 of the 5 antibodies screened.The last antibody did not detect Furin protein under the Western Blotconditions tested. Control lysates provided by the commercial vendorsproduced similar results to in-house cell line samples. The pre-labeledantibody for Flow Cytometry did not demonstrate a significant shift inFurin staining (i.e., no positive Furin population identified).Therefore, the Flow Cytometry could not be used to demonstrate Furinknockdown.

As an alternative to Furin protein detection, the inventors alsoscreened samples for Furin enzyme activity. Using a fluorometric basedassay, cell lines were screened for the conversion of substrate(Pyr-Arg-Thr-Lys-Arg-AMC (SEQ ID NO: 7)) by Furin to release thefluorophore (AMC). However, the detected signal of released AMC was toolow to accurately demonstrate significant knockdown of Furin enzymeactivity. A second barrier to the assay is that the substrate is cleavedby all serine proteases in the subtilisin-like prohormone convertase(PC) family. Therefore, similar proteases that are not targeted by ourFANG shRNA product would remain active and cleave the fluorogenicsubstrate in the assay, thus reducing the capability to detect Furinknockdown.

Other applications for the bi-functional shRNA^(furin) include: (1)Systemic delivery via a tumor (±tumor extracellular matrix (ECM))selective decorated (targeted), stealthed bilamellar invaginatedliposome (BIV) to enhance the efferent limb of the immune response; (2)Systemic delivery via a tumor selective decorated (targeted), stealthedbilamellar invaginated liposome (BIV) to directly subvert the tumorpromoting/maintaining effects of furin target molecules including, butnot limited to human, TGF-β1, TGF-β2, TGF-β3, IGF-II, IGF-1R, PDGF A,and, in some tumor types, MT1-MMP; (3) Systemic delivery via a tumorselective decorated (targeted), stealthed bilamellar invaginatedliposome (BIV) to directly subvert the NOTCH/p300 pathway in putativecancer stem cells; (4) Systemic delivery via a tumor selective decorated(targeted), stealthed bilamellar invaginated liposome (BIV) to inhibitactivation of toxins associated with anthrax, Shiga, diphtheria,tetanus, botulism and Ebola and Marburg viruses; and/or (5) Systemicand/or inhalational delivery of a bilamellar invaginated liposome (BIV)(±decoration and reversible masking/stealthing) to inhibit Pseudomonasexotoxin A production as an adjunct to antibiotic therapy in patientswith diseases with heightened risk of Pseudomonas mediated morbidity andmortality, e.g., cystic fibrosis.

TGF-β Knockdown: Transforming growth factors beta (TGF-β) are a familyof multifunctional proteins with well-known immunosuppressive activities[13]. The three known TGF-β ligands (TGF-β1, TGF-β2, and TGF-β3) areubiquitous in human cancers. TGF-β overexpression correlates with tumorprogression and poor prognosis [14, 15]. Elevated TGF-β levels withinthe tumor microenvironment are linked to an anergic antitumor response[14, 16-21]. TGF-β inhibits GM-CSF induced maturation of DCs [22] andtheir expression of MHC class II and co-stimulatory molecules [23].Ardeshna [24] showed that lipopolysaccharide (LPS)-induced maturation ofmonocyte-derived DCs involved activation of p38 stress-activated proteinkinase (p38SAPK), extracellular signal-regulated protein kinase (ERK),phosphoinositide 3-OH-kinase (PI3 kinase)/Akt, and nuclear factor(NF)-KB pathways. GM-CSF can exert parallel activities of stimulatingmyeloid hematopoietic cell and leukemia cell line proliferation throughrapid, transient phosphorylation of MAP kinase 1/2 and ERK 1/2, whereasTGF-β turns off GM-CSF-induced ERK signaling via PI3-kinase-Akt pathwayinhibition [25].

At the efferent level, antigen presentation by immature DCs contributesto T cell anergy [26]. TGF-β similarly inhibits macrophage activation[27] and their antigen presenting function [28, 29]. TGF-β inhibits theactivation of cytotoxic T cells by impairing high affinity IL-2 receptorexpression and function [30, 31]. TGF-β2 also converts naïve T cells toTreg cells by induction of the transcription factor FOXP3 [32], withemergence of Treg leading to the shutdown of immune activation [33].According to Polak [34], tolerogenic DCs and suppressor T lymphocyteswere present in all stages of melanoma. These immune cell typesexpressed TGF-β receptor I, and tolerogenic activity was dependent onTGF-β1 or -β2 binding.

At the innate immune response level, TGF-β is antagonistic on NK cellsand down-regulates lymphokine activated killer (LAK) cell induction andproliferation [30, 35-39]. Penafuerte [40] recently showed thattumor-secreted TGF-β suppressed GM-CSF+IL2 (GIFT2) mediated immunosensitization of NK cells in the immunocompetent B16 melanoma model. Invivo blockade of B16 production of TGF-β improved survival otherwisecompromised by the growth of non-GIFT2 expressing bystander tumors.These findings further validate the negative impact of TGF-β onGM-CSF-mediated immune activation in vivo, and by extension, support therationale of depleting TGF-β secretion in GM-CSF-based cancer cellvaccines.

Trials conducted by the present inventors utilizing a tumor cell vaccinewith TGF-β2 knockdown activity (Belagenpumatucel-L) in patients withnon-small cell lung cancer demonstrated acceptable safety, and adose-related survival improvement in response to randomized controlpatients and historical experience. The two-year survival for the latestage (IIIB/IV) patients was 52% for patients who received >2.5×10⁷cells/injection, which compares favorably with similar patienthistorical data of less than 10% survival at 2 years. The study patientsalso displayed significantly elevated cytokine production (IFN-γ,p=0.006; IL-6, p=0.004; IL4, p=0.007) and antibody titers to vaccine HLAantigens (p=0.014), suggesting an immune activating outcome [41].

TGF-β-knockdown and GM-CSF Expressive Cancer Cell Vaccine (TAG): Thirtysix patients were harvested for TAG vaccine. GM-CSF expression andTGF-β2 knockdown met product release criteria. Three (allgastrointestinal tumors with luminal access) had bacterial contaminantsand could not be released. One had insufficient cells. Nineteen advancedrefractory cancer patients were treated [42-44]. No Grade 3 toxiceffects related to therapy were observed. Eleven of 17 (65%) evaluablepatients maintained stable disease for at least 3 months. One patientachieved CR by imaging criteria (FIG. 4; melanoma). Thus the TAG vaccineappears to be safe and has evidence of clinical efficacy.

A potential limitation of TAG vaccine, however, is the restrictedspecificity for TGF-β2, given that all three known isoforms of TGF-βligand (TGF-β1, -β2, and -β3) are ubiquitously produced in humancancers. In particular, up to a 10-fold higher level of TGF-β1 may beproduced by human colorectal, lung cancer, and melanoma cells. Thetolerogenic role of TGF-β1 in antigen presenting dendritic cells (DC)and regulatory T cells (Treg) is well established, and this activity isnot impacted by TGF-β2 antisense treatment.

Furin: All mature isoforms of TGF-β require limited proteolytic cleavagefor proper activity. The essential function of proteolytic activation ofTGF-β is mediated by furin. Furin is a member of the subtilisin-likeproprotein convertase family. The members of this family are proproteinconvertases (PCs) that process latent precursor proteins into theirbiologically active products. Furin, a calcium-dependent serineendoprotease, efficiently cleaves precursor proteins at their pairedbasic amino acid processing sites by the consensus sequence-Arg-X-K/Arg-Arg (RXK/RR), (SEQ ID NO: 6), with -RXXR- (SEQ ID NO: 1)constituting the minimal cleavage site [53]. Like many other proteases,PCs are synthesized as inactive zymogens with an N-terminal prosegmentextension, which is autocatalytically removed in the endoplasmicreticulum to achieve functionality [52].

Furin is best known for the functional activation of TGF-β withcorresponding immunoregulatory ramifications [54, 55]. Apart from thepreviously described immunosuppressive activities of tumor secretedTGF-β, conditional deletion of endogenous-expressing furin in Tlymphocytes was found to allow for normal T-cell development, butimpaired the function of regulatory and effector T cells, which producedless TGF-β1. Furin-deficient Tregs were less protective in a T-celltransfer colitis model and failed to induce Foxp3 in normal T cells.Additionally, furin-deficient effector cells were inherently over-activeand were resistant to suppressive activity of wild-type Treg cells. InAPCs, cytotoxic T lymphocyte-sensitive epitopes in the trans-Golgicompartment were processed by furin and the less frequented TAPindependent pathway [56]. Thus furin expression by T cells appears to beindispensable in maintaining peripheral tolerance, which is due, atleast in part, to its non-redundant, essential function in regulatingTGF-β1 production.

High levels of furin have been demonstrated in virtually all cancerlines [45-52]. The present inventors and others have found that up to a10-fold higher level of TGF-β1 may be produced by human colorectal, lungcancer, and melanoma cells, and likely impact the immune tolerance stateby a higher magnitude [34, 57, 58]. The presence of furin in tumor cellslikely contributes significantly to the maintenance of tumor directed,TGF-β1 mediated peripheral immune tolerance [54]. Hence furin knockdownrepresents a novel and attractive approach for optimizingimmunosensitization.

FANG (furin shRNA and GM-CSF) vaccine: The present inventors constructedthe next generation vaccine termed FANG. The novelty of the FANG vaccinelies in the combined approach of depleting multiple immunosuppressiveTGF-β isoforms by furin knockdown, in order to maximize the immuneenhancing effects of the incorporated GM-CSF transgene on autologoustumor antigen sensitization.

All mature isoforms of TGF-β require proteolytic activation by furin.The feasibility of achieving concomitant depletion of multiple TGF-βisoform activity in several cancer cell lines (H460, CCL-247, CRL-1585,U87) was determined using furin-knockdown and the present inventors havesuccessfully completed GMP manufacturing of FANG vaccine in 9 cancerpatients (breast—1; colon—2; melanoma—4; gallbladder—1; NSCLC—1).Assessment of GM-CSF expression and TGF-β1 and -β2 knockdown is shown inFIGS. 1A-1C and FIGS. 2A-2F.

Electroporation of FANG plasmid into patient tumor cells demonstratedGM-CSF protein production and concomitantly TGF-β1 and -β2 knockdown aspredicted. FIGS. 3A-3C depicts Day 7 assay data of a FANG-transfectedNSCLC tumor's expression profile (FANG-004) versus tissue from the samethe tumor processed by the cGMP TAG vaccine method (denoted TAG-004).There are similar reductions in TGF-β2 (FIG. 3B; single line shown dueto coincident data) and similar increases in GM-CSF (FIG. 3C)expression. However, while TGF-β1 expression is completely inhibited byFANG, it is unaffected by TAG as the TGF-β2 antisense cannot blockTGF-β1 expression (FIG. 3A).

FIG. 5 is a PET CT image of an advanced melanoma patient after 11 TAGvaccine treatments demonstrating a significant clinical response.Residual uptake at L 2 was followed up with a MRI scan and biopsy whichrevealed no malignancy. The patient has consequently become a completeresponse (no evidence of disease) for greater than seven months. Thecapability of FANG to knockdown both TGF-β1 and -β2 is supported byfindings in the first 9 patients (FIGS. 6A-6C) who underwent vaccineconstruction. All 9 vaccine preparations demonstrated significantlyelevated levels of GM-CSF (80-1870 pg/ml at day 4 of culture, median of739 pg/ml). All 9 patients demonstrated >50% reductions of TGF-β2, and 6of 7 patients with >100 pg of endogenous TGF-β1 production alsodemonstrated >50% reduction of this cytokine. The expanded targeteffectiveness of FANG is best demonstrated in one patient (NSCLC) whohad adequate tumor tissue to generate both TAG (TAG-004A) and FANG(FANG-004) versions of autologous vaccine, TGF-β1 (as well as TGF-β2)was depleted to below detectable levels using the FANG preparation(FANG-004) from an initial concentration of 1840 pg/ml whereas this highlevel of TGF-β1 was unchanged with the TAG preparation (TAG-004) albeitwith the expected depletion of TGF-β2. These findings support thepotential advantage of the FANG vaccine preparation.

Validation of bioactivity of personalized cGMP FANG vaccines: Genemodification will be achieved by the use of a plasmid vector encodingfor GM-CSF and a bi-functional short hairpin (bi-sh) RNA optimized forfurin knockdown. Cancer patient autologous FANG vaccine has already beengenerated under cGMP conditions for clinical trial of patients withadvanced solid cancers. GM-CSF and TGF-β1, -β2, and -β3 mRNA and proteinexpression were measured as part of the quality assurance process.Cytokine bioactivity following FANG modification was determined bygrowth outcome in a GM-CSF and TGF-β dependent cell line utilized by thepresent inventors in previous studies. Processed vaccine will undergoproteogenomic screening to verify antigenic integrity following FANGmodification.

To characterize the augmenting effect of CTLA-4 blockade: Given thatFANG immunization only impacts the afferent immunosensitization process,additional approaches that promote tumor-specific immune effectorresponses may further promote antitumor outcome. Disrupting Tregsuppression and/or enhancing T effectors (Teff) by blockade of thecytotoxic T lymphocyte-4 (CTLA-4) function may enhance the likelihood ofclinical success of the FANG vaccine.

RT-qPCR analysis was performed on ten FANG vaccine samples (FANG-003 didnot have adequate mRNA for analysis). Samples were culturedpre-electroporation and post-electroporation, post-irradiation for up to14 days. Total RNA was extracted from each sample at various time pointsand converted into cDNA via reverse transcription (RT). Quantitative PCR(qPCR) was performed to assess the amount of template present in eachsample, at each time point. Furin, TGF-β1, and TGF-β2 qPCR samples werenormalized to endogenous GAPDH to produce a relative cycle threshold(Ct) value. GM-CSF was quantified against an external standard curve toproduce an absolute Ct value, relative to the standard curve. The GM-CSFmRNA detection is shown in FIG. 7. Post-transfection, GM-CSF mRNA isdetected in all vaccines but the values are variable depending on mRNAquality—a persistent issue. Table 2 illustrates representative data fromtwo FANG vaccines (FIGS. 8 and 9). All samples were calculated asnormalized pre-electroporation Ct values minus normalizedpost-electroporation, post-irradiation Ct values (pre-post) to calculatethe delta Ct (Δ Ct). A calculated Δ Ct<0.00 represents a decrease intemplate DNA and a calculated Δ Ct>0.00 represents an increase intemplate DNA. The Δ Ct value is used to estimate the percent change inexpression (% expression). Values less than 100% represent a decrease inDNA (from pre to post) and values greater than 100% represent anincrease in DNA (from pre to post). The nature of shRNA/siRNA silencingcan optimally reduce the template DNA 90%, which is equivalent to a ΔCt=−3.3. (A Δ Ct=−1.0 is equivalent to a 50% knockdown.) Therefore, thedata below demonstrate that the FANG plasmid DNA is able to reduceendogenous Furin down 80-26% (average=48%) and the downstream targetsTGF-β1 and TGF-β2 are reduced down 98-30% (average=75%). The mechanismsof action of the Furin bi-functional shRNA are to block Furin proteinproduction at the post-transcriptional and translational levels. Thereduced levels of Furin protein also impact (by feedback regulation) theexpression of TGF-β1 and TGF-β2 mRNA, the conversion of the proform ofTGF-β1 and TGF-β2 protein into the mature (active) form of theirrespective proteins [75], and, by interfering with the TGFβ→furinamplification loop, further dampen the expression of furin itself [76].It is also possible that accumulation of the proform of the TGF proteinmay feedback inhibit the transcription of its TGF gene.

TABLE 2 RT-qPCR Analysis of FANG Vaccines (Pre Versus PostElectroporation) FANG-008 Time Point Δ Ct % Expression Furin day 0 −1.5235% day 1 −1.50 35% day 2 −1.48 36% day 4 −1.50 35% day 7 −1.22 43% day10 −1.41 38% TGFB1 day 0 −0.09 94% day 1 −0.10 93% day 2 −0.08 95% day 4−0.05 97% day 7 −0.08 95% day 10 −0.11 93% TGFB2 day 0 0.00 n/a * day 10.00 n/a * day 2 0.00 n/a * day 4 0.00 n/a * day 7 0.00 n/a * day 100.00 n/a * FANG-009 Ident. Δ Ct % Expression Furin day 0 −0.66 63% day 1−0.69 62% day 2 −0.34 79% day 4 −0.31 80% day 7 −1.93 26% day 10 0.00n/a * TGFB1 day 0 −0.54 69% day 1 −0.49 71% day 2 −0.42 75% day 4 −0.3181% day 7 −0.04 98% day 10 −1.29 41% TGFB2 day 0 −0.53 69% day 1 −0.4772% day 2 −0.45 73% day 4 −0.52 70% day 7 −1.70 31% day 10 −1.74 30% ΔCt baseline = 0.00 % expression baseline = 100% * n/a = not applicablebecause template was below detection limits

The FANG system was used with 9 patient autologous vaccines, whichconsistently demonstrated TGF-β1 and TGF-β2 reductions and elevatedGM-CSF levels (FIGS. 6A-6C). Both TGF-β1 and TGF-β2 activity by specificimmunoassay was also demonstrated to be significantly reduced in thesecancer lines, confirming the effect of furin blockade on TGF-β isoformexpression. The inventors validated the applicability of siRNA-mediatedfurin-knockdown for inhibiting TGF-β isoform expression. ProspectivesiRNA targeting sites in the furin mRNA sequence (FIG. 4B) weredetermined by the published recommendations of Tusch1 and colleagues andthe additional selection parameters that integrated BLAST searches ofthe human and mouse genome databases. siRNAs targeting eligibletranslated and 3′ UTRs sites (FIG. 4B) were tested. Demonstration ofFANG plasmid DNA knockdown of furin mRNA is shown in FIGS. 8 and 9. Thiscould only be detected in two of the vaccines because readily detectablefurin mRNA was present in only these two tumors pretransfection. Themechanisms of action of the bi-shRNA^(furin) are the blockade of furinprotein production at the post transcriptional and translational levels.The reduced levels of furin protein also impact (by feedback regulation)the expression of TGF-β1 and TGF-β2 mRNA, the conversion of the proformTGF-β1 and TGF-β2 protein into the mature (active) form of theirrespective proteins [75] and by interfering with the TGF-β→furin loop,further dampening the expression of furin itself [76]. The possibilitythat the accumulation of the pro form of the TGF protein may feedbackand inhibit the transcription of its TGF gene should not be in any wayconstrued as a limitation of the present invention. The expanded targeteffectiveness of FANG is best demonstrated in one patient (NSCLC) whohad adequate tumor tissue to generate both TAG (TAG-004) and FANG(FANG-004) versions of autologous vaccine. TGF-β1 (as well as TGF-β2)was depleted to below detectable levels using the FANG preparation(FANG-004) from an initial concentration of 1840 pg/ml. This high levelof TGF-β1 was unchanged with the TAG preparation (TAG-004) albeit withthe expected depletion of TGF-β2 (FIGS. 1A-1C and FIGS. 2A-2F). Thesefindings support the mechanistic advantage of the FANG vaccinepreparation.

Following lipofection of CCL-247, CRL-1585 U87 and H460 cells, each ofthe 6 siRNA^(furin) constructs was shown to markedly reduce TGF-β1 andTGF-β2 levels in culture supernatants without adversely affecting cellsurvival. Thus siRNA-mediated furin knockdown is effective for thedepletion of TGF-β1 and -β2 isoforms.

Design and construction of FANG: A “bi-functional” vector was used thatincorporates both siRNA and miRNA-like functional components foroptimizing gene knockdown [61]. The siRNA component is encoded as ahairpin and encompasses complete matching sequences of the passenger andguide strands. Following cleavage of the passenger strand by theArgonaute-2 (Ago 2) of the RNA-induced silencing complex (RISC), anendonuclease with RNase H like activity, the guide strand binds to andcleaves the complementary target mRNA sequence. In distinction, themiRNA-like component of the “bi-functional” vector incorporatesmismatches between the passenger and guide strands within the encodingshRNA hairpin in order to achieve lower thermodynamic stability. Thisconfiguration allows the passenger strand to dissociate from RISCwithout cleavage (cleavage-independent process) independent of Ago 2[62, 63], and the miRNA guide component to downregulate its targetthrough translational repression, mRNA degradation, and sequestration ofthe partially complementary target mRNA in the cytoplasmic processingbodies (P-body).

The inventors have previously demonstrated the enhanced effectiveness ofa bi-functional shRNA to knockdown stathmin (STMN1; oncoprotein 18), aprotein that regulates rapid microtubule remodeling of the cytoskeletonand found to be upregulated in a high proportion of patients with solidcancers [64]. The bi-functional shRNA construct achieved effectiveknockdown against STMN-1 resulting in a 5-log dose enhanced potency oftumor cell killing as compared with siRNA oligonucleotides directedagainst the same gene target.

A similarly designed bi-functional shRNA was used to effect furinknockdown. The bi-sh-furin consists of two stem-loop structures with amiR-30a backbone; the first stem-loop structure has completecomplementary guiding strand and passenger strand, while the secondstem-loop structure has three bp mismatches at positions 9 to 11 of thepassenger strand. The inventors adopted a strategy of using a singletargeted site for both cleavage and sequestration processes. Theencoding shRNAs are proposed to allow mature shRNA to be loaded ontomore than one type of RISC [65]. The inventors focused on a single sitesince multi-site targeting may increase the chance for “seed sequence”induced off-target effects [66].

The two stem-loop structure was put together with 10 pieces ofcomplementing an interconnecting oligonucleotides through DNA ligation.Orientation of the inserted DNA was screened by PCR primer pairsdesigned to screen for the shRNA insert and orientation. Positive cloneswere selected and sequence confirmed at SeqWright, Inc. (Houston, Tex.).Based on siRNA findings, three bi-functional shRNAs were constructed.The optimal targeting sequence was identified.

The FANG construct has a single mammalian promoter (CMV) that drives theentire cassette, with an intervening 2A ribosomal skip peptide betweenthe GM-CSF and the furin bi-functional shRNA transcripts, followed by arabbit polyA tail. There is a stop codon at the end of the GM-CSFtranscript.

Insertion of picornaviral 2A sequences into mRNAs causes ribosomes toskip formation of a peptide bond at the junction of the 2A anddownstream sequences, leading to the production of two proteins from asingle open reading frame [67]. The inventors found that the 2A linkerto be effective for generating approximately equal levels of GM-CSF andanti-TGF-β transcripts with the TAG vaccine, and elected to use the samedesign for FANG.

Manufacturing the FANG vaccine: The patient's tumor was asepticallycollected in the surgical field, placed in a gentamycin saline solutionin a sterile specimen container and packaged for shipment on wet ice tothe cGMP manufacturing facility. The specimen was brought into themanufacturing suite, dissected, enzymatically and mechanicallydisaggregated to form a cell suspension and then washed to removedebris. After the tumor cells are enumerated, QC aliquots are taken andthe remaining cells are electroporated with the FANG plasmid andincubated overnight to allow vector transgene expression. Cells areharvested and gamma irradiated to arrest cell growth, then enumeratedprior to removal of final QC aliquots and vaccine controlled ratefreezing. The two day manufacturing process was followed by an almostthree week QC testing phase after which all of vaccine assay data areevaluated prior to releasing the vaccine for patient treatment. All 9initial patients who underwent FANG manufacturing passed all QC testingcriteria.

cGMP FANG vaccines: Cancer patient autologous FANG vaccines weregenerated under cGMP conditions for use in clinical trials. GM-CSF andTGF-β1, -β2, and -β3 mRNA and protein expression were measured beforeand after FANG modification, and cytokine bioactivity determined bygrowth outcome on a GM-CSF and TGF-β dependent human cell line we havepreviously characterized. Each patient's processed vaccine will undergoproteogenomic screening to verify antigenic integrity following FANGmodification.

cGMP production of FANG: FANG vaccine was generated by plasmid vectorelectroporation of established human cell lines. The selected FANGplasmid vector represents a construct containing the furin shRNA thathas been prevalidated for optimal TGF-β downregulation.

Before being injected into patients, a frozen vial (dose) was thawed atroom temperature and processed in a biosafety hood. The cell suspensionwill be delivered in a capped 1 mL syringe. The prepared vaccine will beinjected intradermally into patient at a dose of 1×10⁷ or 2.5×10⁷ cellsper injection.

Two full scale preclinical manufacturing processes and eight clinicalmanufacturing processes were prepared and studies by the presentinventors. Table 3 depicts the types of tumors processed (tumors 3through 10 are the clinical vaccines).

TABLE 3 Tumor mass versus cell yield. Tumor Tissue Weight Cell #/Processed Vaccine ID (grams) dose Number of Vials 1 FANG-001 12.72 1.0 ×10⁷ 40 2 FANG-002 27.41 1.0 × 10⁷ 28 3 FANG-003 6.04 2.5 × 10⁷ 9 4FANG-004 41.08 2.5 × 10⁷ 11 5 FANG-005 6.96 2.5 × 10⁷ 8 6 FANG-006 12.481.0 × 10⁷ 8 7 FANG-007 10.90 2.5 × 10⁷ 15 8 FANG-008 9.80 2.5 × 10⁷ 13 9FANG-009 6.80 1.0 × 10⁷ 6 10 FANG-010 13.00 2.5 × 10⁷ 12

The tumors processed range in size, as well as type, and the resultingviable cell yield varies greatly as shown in Table 4. All vaccines arevialed at either 1.0×10⁷ cells (dose Cohort 1) or 2.5×10⁷ cells (doseCohort 2) depending on the total viable cell count on Day 2 ofmanufacturing. Patients with multiple tumor harvests were allowed tocombine vials to qualify for minimum clinical dose requirement. Amaximum of 12 doses at Cohort 2 dose level will be made available forpatient treatment. Because tumor cell yield is highly variable due totumor mass, cellularity, and processing compatibility, the minimum dosenumber and lower dose cohort (Cohort 1) were included.

TABLE 4 Final product viability (Day 2, Pre Irradiation) Tumor ProcessedVaccine ID % Viability 1 FANG-001 78 2 FANG-002 90 3 FANG-003 94 4FANG-004 89 5 FANG-005 94 6 FANG-006 91 7 FANG-007 96 8 FANG-008 95 9FANG-009 95 10 FANG-010 93

The Day 4 expression profiles of the 10 tumors processed are depicted inFIGS. 1A-1C. Note that they-axis scales are different for all threecytokines. These data are representative of the 14 day assay (remainderof data not shown). The mean pre-transfection TGF-β1 is 1251±1544pg/1×10⁶ cells/ml; median 778 pg. The mean post-transfection TGF-β1 is191±455 pg/lx 10⁶ cells/ml; median 13 pg. The average percent knockdownof TGF-β1 was 85%. The mean pre-transfection TGF-β2 is 232±164 pg/1×10⁶cells/ml; median 225 pg. The mean post-transfection TGF-β2 is 1319pg/1×10⁶ cells/ml; median 5 pg. The average percent knockdown of TGF-β2was 94%. The average GM-CSF expression post transfection is 543±540pg/1×10⁶ cells/ml; median 400 pg. These data indicate that the GM-CSFexpression is consistent with the TAG vaccine as is the TGF-β2knockdown. In contrast, FANG vaccines have reduced the TGF-β1 expressionmore than fivefold. The minimum detectable quantity of TGF-β1 isapproximately 4.6 pg/ml (R&D Systems, Quantikine Human TGF-β1). Theminimum detectable quantity of TGF-β2 is approximately 7 pg/ml (R&DSystems, Quantikine Human TGF-β2). The minimum detectable quantity ofGM-CSF is approximately 3 pg/ml (R&D Systems, Quantikine Human GM-CSF).

The protocol for setting up cultures pre and post Transfection forAutologous tumor cell vaccine to test for the expression of GM-CSF,TGF-β1 and TGF-β2 has been previously described (Maples, et al., 2009).Briefly, GMCSF, TGF-β1 and TGF-β2 expression were determined bycommercially available ELISA kits (R & D Systems). The ELISA assays wereperformed according to manufacturer's instruction. The pre-transfectionsample (4×10⁶ cells) is taken on Day 1. After manufacturing iscompleted, the sample is removed from the manufacturing facility so thatthe cell cultures can be set up for generating the sample for ELISA. OnDay 2, the post-transfection, post-irradiation, pre-freeze sample (4×10⁶cells) is taken. After manufacturing is completed, the sample is removedfrom the manufacturing facility so that the cell cultures can be set upfor generating the sample for ELISA.

Ten (10) vaccines (FANG-001 to -010) have been manufactured as part ofthe preclinical qualification process. These vaccines have beenevaluated for GM-CSF, TGF-β1 and TGF-β2 mRNA and protein expressionusing post-transfection, post-irradiation samples compared withpre-transfection, pre-irradiation samples (per FDA review, TAG vaccine,BB-IND 13650). In addition, Furin protein detection was attempted byseveral methods. Furin mRNA was detected by qRT-PCR.

The present inventors detected endogenous Furin protein in cell linesvia Western Blot and Flow Cytometry. Five (5) different antibodies (from3 different vendors) were screened for Western Blot and one (1)pre-labeled antibody was screened for Flow Cytometry. All experimentsyielded negative results (data not shown).

A summary of all ELISA data for all manufacturing processes (Table 5)indicates that the median level of GM-CSF expression is about 400picogram/ml and the average is 543 picograms/ml. Further, the level ofGM-CSF tends to increase with time. In all manufactured products, GM-CSFexpression is observed although the level of expression is variablebetween manufacturing processes (tumor types). In addition to documentedvariability in the level of GM-CSF expression between manufacturingprocesses, the levels of expression achieved with the FANG vaccine aredeemed clinically relevant as 1) use of a plasmid rather than a viralvector obviates the obfuscating effects of elicited anti-viralneutralizing antibodies, 2) use of a plasmid likewise prevents thedevelopment of elicited antibodies interfering with long-term geneexpression, and 3) concurrent suppression of furin, TGF-β1, and TGF-β2will minimize tumor associated inhibition of GM-CSF induced dendriticcell maturation [25].

TABLE 5 FANG vaccines 1-10 TGF-β1, TGF-β2 and GM-CSF expression in the14 Day post manufacturing expression assay. TGF-β1 TGF-β1 TGF-β2 TGF-β2pg/ml Pre pg/ml Post pg/ml Pre pg/ml Post Mean SD Median Mean SD MedianMean SD Median Mean SD Median Day 0 625 678 416 105 202 7 70 116 25 9 220 Day 1 1154 1266 760 93 187 11 138 139 113 9 19 0 Day 2 998 1014 620180 446 0 199 107 197 12 21 4 Day 3 1832 3221 879 173 394 4 247 156 22912 16 8 Day 4 1241 1115 1039 211 421 20 293 189 257 9 12 4 Day 7 17291735 778 264 723 3 292 150 235 14 16 8 Day 10 1367 994 1629 243 530 21335 135 310 23 21 28 Day 14 1108 892 887 281 661 19 308 158 229 17 23 12Overall 1251 1544 778 191 455 13 232 164 225 13 19 5 GM-CSF GM-CSF pg/mlPre pg/ml Post Mean SD Median Mean SD Median Day 0 2 2 2 157 277 29 Day1 3 4 3 359 469 281 Day 2 3 3 3 407 418 310 Day 3 3 4 2 580 531 475 Day4 4 6 3 657 550 602 Day 7 5 9 3 683 681 471 Day 10 5 8 4 745 546 673 Day14 18 24 4 821 631 645 Overall 5 10 3 543 540 400

Quantification of GM-CSF and TGF-β expressions: GM-CSF and TGF-β1 and-β2 expression was determined by cytokine specific colorimetric assay[68].

Validation of bioactivity: GM-CSF-induced proliferative activity similarto that of myeloid hematopoietic cells has been observed in myeloidleukemia cell lines, as mediated by the rapid and transientphosphorylation of MAP kinase 1/2 and ERK 1/2. By contrast, TGF-β turnsoff GM-CSF-mediated ERK signaling by inhibition of the PI3-kinase-Aktpathway [25]. The growth regulatory effects of GM-CSF and TGF-β onmyeloid leukemia cells were used as an in vitro surrogate model tovalidate cytokine bioactivity in prepared FANG vaccine culturesupernatants.

Cytokine activities in the FANG (or control-transfected) vaccine culturesupernatants were validated by co-culture studies with erythroleukemiaCD34+TF-1a cells [69] and, if necessary, confirmed with the biphenotypicB myelomonocytic leukemia CD10+CD15+MV4-11 cells [70] (ATCC, Rockville,Md.). Both of these cell lines have been shown respond to the positiveproliferative effects of GM-CSF and the negative inhibitory activity ofTGF-β at ng/ml amounts [25]. Proliferative activity will be determinedby Easycount Viasure assay (Immunicon) and MTT assay [68].

Phenotypic profile analysis of FANG modification: Furin knockdown likelyimpacts the expression of other protein substrates with the targetsequence in addition to TGF-β downregulation [51]. The antigenic profileof the FANG-processed autologous vaccines were determined from cancerpatients, in the event that this information may be useful towards theunderstanding any differential clinical outcome in vaccinated patients.

High throughput genetic profiling was used to develop individualizedtherapeutics for cancer patients. High throughput, gene expression arrayanalysis was carried out to compare the differential gene expressionprofile of FANG-transfected vs. control vector-transfected cancer cells.

Differentially labeled FANG and control preparations are combined andfractionated by high performance liquid chromatography (Dionex), using astrong cation exchange column (SCX) and a 2^(nd) dimension RP nanocolumn. The fractions are spotted onto Opti-TOF™ LC/MALDI Insert (123×81mm) plates (Applied Biosystems) in preparation for mass spectrometryanalysis using the Applied Biosystems 4800 MALDI TOF/TOF™ Analyzer. Bothprotein and gene expression data were then evaluated by the GeneGo,MetaCore software suite.

Proteogenomic analysis was carried out for the purpose of determiningthe antigen repertoire of the autologous cancer vaccine before and afterFANG process. In addition to the validation of furin knockdown,particular attention was focused on 1) baseline and differentialexpression of furin-substrate proteins; 2) expression of landmarktumor-associated antigens (TAAs; such as gp100, Mart1, MAGE-1,tyrosinase, for melanoma; MAGE-3, MUC-1 for non-small cell lung cancer)[71, 72] and other reported TAAs; 3) HLA antigens and co-stimulatorymolecules (CD80/86) expression; 4) proteins unrelated to the abovecategories that are differentially expressed by 2-fold or higherfollowing FANG transfection.

FIG. 10 shows the overall survival for Cohort 1 versus Cohorts 2 and 3for advanced-stage patients (n=61; P=0.0186). A schematic diagram ofGM-CSF-TGF-β2 antisense plasmid is shown in FIG. 11. The expression ofGM-CSF in NCI-H-460 Squamous Cell and NCI-H-520, Large Cell (NSCLC)containing the pUMVC3-GM-CSF-2A-TGF-β2 antisense vector, in vitro isdepicted in FIG. 12. TGF-β2 levels are reduced in NCI-H-460 SquamousCell and NCI-H-520, Large Cell (NSCLC) with the pUMVC3-GM-CSF-2A-TGF-β2antisense vector. This reduction is seen in the date presented in FIG.13. FIG. 14 shows that a 251 base pair probe specifically detects theGM-CSF-2A-TGF-β2 transcript expressed in vitro in NCI-H-460 andNCI-H-520 cells (lanes 6 and 10); GM-CSF and TGF-β2 expression in TAGvaccines are shown in FIGS. 12 and 13, respectively. Expression ofTGF-β1 and TGF-β2 in human cancer lines following siRNA^(furin)knockdown is shown in FIGS. 18A and 18B. Finally FIG. 19 is a schematicof the plasmid construct of FANG.

TGF-β1 expression data (FIG. 16) generated from TAG vaccinemanufacturing data (n=33 vaccines; Day 7 values, TGF-β1 assay postvaccine manufacturing) clearly demonstrate that TAG does not interferewith TGF-β1 expression. The clinical significance of blocking TGF-β1 andTGF-β2 (FIG. 17), as well as TGF-β3 (data not shown) is that they arepostulated to be significant negative immunomodulators expressed by thetumor. GM-CSF expression in TAG vaccines is shown in FIG. 15. TheseTGF-β isoforms are ubiquitous and expressed in the majority of tumors[77]. Many tumors, including breast, colon, esophageal, gastric,hepatocellular, pancreatic, SCLC and NSCLC produce high levels of one ormore active TGF-β isoforms [78, 14, 15, 79-84]. Furthermore,overexpression of TGF-β has been correlated with tumor progression andpoor prognosis [14, 15]. Elevated TGF-β levels have also been linkedwith immunosuppression in both afferent efferent limbs [14, 16-21].Additionally, TGF-β has antagonistic effects on Natural Killer (NK)cells as well as the induction and proliferation of lymphokine-activatedkiller (LAK) cells [30, 35-39].

The immune suppressor functions of TGF-β are therefore likely to play amajor role in modulating the effectiveness of cancer cell vaccines.TGF-β inhibits GMCSF induced maturation of bone marrow derived dendriticcells (DCs) [22] as well as expression of MHC class II and costimulatorymolecules [23]. It has been shown that antigen presentation by immatureDCs result in T cell unresponsiveness [26]. TGF-β also inhibitsactivated macrophages [27] including their antigen presenting function[28, 29]. Hence both the ubiquity of expression as well as theinhibitory effects of TGF-β on GMCSF immunomodulatory function supportthe knockdown of all tumor TGF-β expression in the autologous cancervaccine treatment approach of the present invention.

The immune suppressor functions of TGF-β are therefore likely to play amajor role in modulating the effectiveness of cancer cell vaccines.TGF-β inhibits GM-CSF induced maturation of bone marrow deriveddendritic cells (DCs) [25] as well as expression of MHC class II andcostimulatory molecules [26]. It has been shown that antigenpresentation by immature DCs result in T cell unresponsiveness [27].TGF-β also inhibits activated macrophages [28] including their antigenpresenting function [29, 30]. Hence both the ubiquity of expression aswell as the inhibitory effects of TGF-β on GM-CSF immunomodulatoryfunction support the knockdown of all tumor TGF-β expression in thisautologous cancer vaccine treatment approach.

It will be understood that particular embodiments described herein areshown by way of illustration and not as limitations of the invention.The principal features of this invention can be employed in variousembodiments without departing from the scope of the invention. Thoseskilled in the art will recognize, or be able to ascertain using no morethan routine experimentation, numerous equivalents to the specificprocedures described herein. Such equivalents are considered to bewithin the scope of this invention and are covered by the claims.

All publications and patent applications mentioned in the specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.” The use of the term “or” in the claims isused to mean “and/or” unless explicitly indicated to refer toalternatives only or the alternatives are mutually exclusive, althoughthe disclosure supports a definition that refers to only alternativesand “and/or.” Throughout this application, the term “about” is used toindicate that a value includes the inherent variation of error for thedevice, the method being employed to determine the value, or thevariation that exists among the study subjects.

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open--ended and do not exclude additional, unrecitedelements or method steps.

The term “or combinations thereof” as used herein refers to allpermutations and combinations of the listed items preceding the term.For example, “A, B, C, or combinations thereof” is intended to includeat least one of: A, B, C, AB, AC, BC, or ABC, and if order is importantin a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.Continuing with this example, expressly included are combinations thatcontain repeats of one or more item or term, such as BB, AAA, AB, BBC,AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan willunderstand that typically there is no limit on the number of items orterms in any combination, unless otherwise apparent from the context.

As used herein, words of approximation such as, without limitation,“about”, “substantial” or “substantially” refers to a condition thatwhen so modified is understood to not necessarily be absolute or perfectbut would be considered close enough to those of ordinary skill in theart to warrant designating the condition as being present. The extent towhich the description may vary will depend on how great a change can beinstituted and still have one of ordinary skilled in the art recognizethe modified feature as still having the required characteristics andcapabilities of the unmodified feature. In general, but subject to thepreceding discussion, a numerical value herein that is modified by aword of approximation such as “about” may vary from the stated value byat least ±1, 2, 3, 4, 5, 6, 7, 10, 12 or 15%.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

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What is claimed is:
 1. A method of manufacturing abi-shRNA^(furin)/GMCSF cancer vaccine, comprising: (a) forming asuspension of tumor cells; (b) transfecting the tumor cells with abi-shRNA^(furin)/GMCSF expression vector plasmid comprising (i) a firstinsert comprising a nucleic acid sequence encoding a human GranulocyteMacrophage Colony Stimulating Factor (GM-CSF) cDNA; and (ii) a secondinsert comprising a nucleic acid sequence encoding a bi-functional shorthairpin RNA (bi-shRNA) capable of hybridizing to a furin mRNAtranscript; (c) harvesting the transfected tumor cells; and (d) freezingthe transfected tumor cells.
 2. The method of claim 1, furthercomprising harvesting a tumor comprising tumor cells from an individualand placing the tumor in an antibiotic solution in a sterile containerprior to the formation of the tumor cell suspension.
 3. The method ofclaim 1, wherein the tumor cell suspension is formed by enzymaticdissection, mechanical disaggregation, or any combination thereof. 4.The method of claim 1, wherein the tumor cells are transfected byelectroporation with the expression vector.
 5. The method of claim 1,further comprising incubating the transfected tumor cells overnightprior to harvesting.
 6. The method of claim 1, further comprisingrendering the transfected tumor cells proliferation-incompetent prior tofreezing.
 7. The method of claim 6, wherein the transfected tumor cellsare rendered proliferation-incompetent by irradiation.
 8. The method ofclaim 6, wherein the transfected tumor cells are renderedproliferation-incompetent by X-ray irradiation.
 9. The method of claim1, wherein the transfected tumor cells are enumerated and aliquotedprior to freezing.
 10. The method of claim 1, wherein the tumor cellsare derived from a melanoma, a non-small-cell lung cancer, a gallbladder cancer, a colorectal cancer, a breast cancer, a ovarian cancer,a liver cancer, or a Ewing's sarcoma.
 11. The method of claim 1, furthercomprising incubating the transfected tumor cells with γIFN aftertransfection.
 12. The method of claim 11, wherein the transfected tumorcells are incubated with about 100 U/ml of γIFN for 48 hours or about500 U/ml of γIFN for 24 hours.
 13. The method of claim 1, wherein thefirst insert is operably linked to a promoter.
 14. The method of claim13, wherein the second insert is operably linked to the promoter. 15.The method of claim 13, wherein the promoter is a CMV mammalian promoterand the expression vector further comprises a CMV IE 5′ UTR enhancersequence and a CMV IE Intron A sequence.
 16. The method of claim 1,wherein the expression vector further comprises a picornaviral 2Aribosomal skip peptide sequence between the first and the second nucleicacid inserts.
 17. The method of claim 1, wherein the bi-shRNA is capableof hybridizing within the 3′ UTR region of the furin mRNA transcript.18. The method of claim 1, wherein second insert comprises: (a) a firststem loop structure comprising (i) a first guide sequence capable ofhybridizing to a furin mRNA transcript; and (ii) a first passengersequence fully complementary to the first guide strand; and (b) a secondstem loop structure comprising (i) a second guide sequence capable ofhybridizing to a furin mRNA transcript; and (ii) a second passengersequence partially complementary to the second guide strand.
 19. Themethod of claim 18, wherein the second passenger sequence has a threebasepair mismatch with the second guide sequence at positions 9 to 11 ofthe second passenger strand.
 20. The method of claim 19, wherein thesecond insert comprises a nucleic acid sequence encoding a bi-functionalshort hairpin RNA (bi-shRNA) according to SEQ ID NO: 2.